The peroxisomal protein import machinery--a case report of transient ubiquitination with a new flavor.

The peroxisomal protein import machinery displays remarkable properties. Be it its capacity to accept already folded proteins as substrates, its complex architecture or its energetics, almost every aspect of this machinery seems unique. The list of unusual properties is still growing as shown by the recent finding that one of its central components, Pex5p, is transiently monoubiquitinated at a cysteine residue. However, the data gathered in recent years also suggest that the peroxisomal import machinery is not that exclusive and similarities with p97/Cdc48-mediated processes and with multisubunit RING-E3 ligases are starting to emerge. Here, we discuss these data trying to distill the principles by which this complex machinery operates.

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation.

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.

Characterization of the mammalian peroxisomal import machinery: Pex2p, Pex5p, Pex12p, and Pex14p are subunits of the same protein assembly.

Although many of the proteins involved in the biogenesis of the mammalian peroxisome have already been identified, our knowledge of the architecture of all this machinery is still very limited. In this work we used native gel electrophoresis and sucrose gradient sedimentation analysis in combination with immunoprecipitation experiments to address this issue. After solubilization of rat liver peroxisomes with the mild detergent digitonin, comigration of Pex5p, Pex14p, and a fraction of Pex12p was observed upon native electrophoresis and sucrose gradient sedimentation. The existence of a complex comprising Pex2p, Pex5p, Pex12p, and Pex14p was demonstrated by preparative coimmunoprecipitation experiments using an antibody directed to Pex14p. No stoichiometric amounts of Pex13p were detected in the Pex2p-Pex5p-Pex12p-Pex14p complex, although the presence of a small fraction of Pex13p in this complex could be demonstrated by Western blot analysis. Pex13p is also a component of a high molecular mass complex. Strikingly, partial purification of this Pex13p-containing complex revealed Pex13p as the major (if not the only) component. Taken together, our data indicate that Pex2p, Pex5p, Pex12p, and Pex14p, on one side, and Pex13p, on the other, are subunits of two stable protein complexes that probably interact with each other in the peroxisomal membrane.

Characterisation of two mutations in the ABCD1 gene leading to low levels of normal ALDP.

A variety of mutations have been identified in the X-linked adrenoleukodystrophy (X-ALD) gene, none of which is prevalent. In this work we describe a reverse transcription polymerase chain reaction (RT-PCR)-based strategy specially suited to the molecular characterisation of mutations in index cases. After RT-PCR amplification of the X-ALD transcript a conformation-sensitive gel electrophoresis analysis is performed followed by sequencing of the fragments with altered mobility. Two X-ALD patients were studied using this strategy. In both cases, splice site mutations were found. The first patient studied has a single base substitution at the first position of the invariant GT dinucleotide donor splice site of intron 8. In spite of this alteration, small quantities of correctly spliced mRNA molecules were easily detected. In agreement with these data, a small amount of ALDP was found by western blotting analysis. An alteration at the -1 position of the donor splice site of exon 1 was detected in the second patient. This mutation results in the utilisation of a cryptic 5' splice site within intron 1. Nevertheless, this transition also allows for some correct splicing. Western blotting analysis revealed the existence of normal-migrating ALDP. However, as expected, the levels of this protein were greatly decreased. Taken together, our data suggest that some less severe or late-onset forms of X-ALD associated with splice mutations result from the production of small amounts of normal ALDP. It is proposed that the quantification of ALDP levels in these patients could provide important insights concerning the correlation between clinical phenotype and amount of normal ALDP.

Characterization of peroxisomal Pex5p from rat liver. Pex5p in the Pex5p-Pex14p membrane complex is a transmembrane protein.

Pex5p is the receptor for the vast majority of peroxisomal matrix proteins. Here, we show that about 15% of rat liver Pex5p is found in the peroxisomal fraction representing 0.06% of total peroxisomal protein. This population of Pex5p displays all the characteristics of an intrinsic membrane protein. Protease protection assays indicate that this pool of Pex5p has domains exposed on both sides of the peroxisomal membrane. The strong interaction of Pex5p with the membrane of the organelle is not affected by mild protease treatment of intact organelles, conditions that result in the partial degradation of Pex13p. Cytosolic Pex5p is a monomeric protein. In contrast, virtually all peroxisomal Pex5p was found to be part of a stable 250-kDa protein assembly. This complex was isolated and shown to comprise just two subunits, Pex5p and Pex14p.

Alkaline density gradient floatation of membranes: polypeptide composition of the mammalian peroxisomal membrane.

A method for purification of the peroxisomal membrane from rat liver is described. The procedure consists of floating the (contaminated) peroxisomal membranes through an alkaline sucrose density gradient. A good resolution between the peroxisomal membrane and other membrane systems is achieved. Using these floated peroxisomal membranes we have determined that only 7.8 +/- 0.9% of the total peroxisomal protein is alkali resistant. The polypeptide composition of these highly pure peroxisomal membranes was analyzed by SDS-PAGE. Bands corresponding to polypeptides with apparent molecular masses of 15, 18, 22, 24, 26, 29, 35, 36, 38, 40, 52, 55, 70, 74-77, and 88 kDa are detected upon Coomassie blue staining of polyacrylamide gels. The identity of several of these polypeptides was determined by N-terminal sequencing and Western blotting analysis.

Identification of a 24 kDa intrinsic membrane protein from mammalian peroxisomes.

A 24 kDa protein from rat liver peroxisomal membrane was isolated and subjected to Edman degradation. Using the N-terminal sequence of this polypeptide we have identified several rat and human expressed sequence tags in the GenBank Database. The complete sequence of a human cDNA clone was determined. The open reading frame encodes an extremely basic protein 212 amino acid residues long. A high similarity between this mammalian protein and hypothetical proteins from Caenorhabditis elegans and Neurospora crassa was found. Hydropathy analysis reveals the existence of two putative membrane-spanning domains in conserved regions of the three homologous proteins.

[Acute hemodynamic effects of ibopamine, captopril, and placebo in patients with severe heart failure]. / Efeitos hemodinâmicos agudos da ibopamina, captopril e placebo em pacientes com insuficiência cardíaca grave.

PURPOSE: To evaluate the acute hemodynamic effects of ibopamine (IBO), captopril (CAP) and placebo (PLA) in patients with severe congestive heart failure at rest. METHODS: Twelve male patients in sinus rhythm with dilated cardiomyopathy and NYHA class IV were studied with Swan-Ganz hemodynamics. Drugs were given in a blinded fashion. Rest, 30 min and every hour for 5 h measurements were made after oral ingestion of 100 mg IBO, 25 mg CAP or PLA. Prior to the study, patients were on diuretics as the only medication for at least 48 h. Comparisons were made with analysis of variance of repeated measurements and Duncan's multiple comparisons procedure. RESULTS: Significant increase in cardiac index and stroke volume index and reduction in systemic vascular resistance were observed with IBO and CAP for 2 h after ingestion. IBO however increased right and left filling pressures in the first hour after its administration. Ventricular tachycardia occurred in 2 patients 1 h after IBO administration. CONCLUSION: Both IBO and CAP improved hemodynamic parameters in the first two hours after oral ingestion in patients with dilated cardiomyopathy in class IV.

Disruption of the gene encoding the 78-kilodalton subunit of the peripheral arm of complex I in Neurospora crassa by repeat induced point mutation (RIP).

We have used the procedure of sheltered RIP to generate mutants of the 78-kDa protein of the peripheral arm of Neurospora crassa complex I. The nuclei containing the mutations were initially isolated as one component of a heterokaryon but subsequent analysis showed that nuclei containing null alleles of the gene could be propagated as homokaryons. This demonstrates that the gene does not serve an essential function. Sequence analysis of one allele shows that 61 transition mutations were created resulting in 39 amino-acid changes including the introduction of four stop codons. Mutant strains grow at a slower rate than wild-type and exhibit a decrease in the production of conidia. Electron paramagnetic spectroscopy of mutant mitochondria suggest that they are deficient in Fe-S clusters N-1, N-3, and N-4.

Two nuclear-coded subunits of mitochondrial complex I are similar to different domains of a bacterial formate hydrogenlyase subunit.

A computer comparison of protein sequences revealed similarity between the 30.4 kDa subunit of complex I from the fungus Neurospora crassa and the ORF5 subunit of formate hydrogenlyase from Escherichia coli. The ORF5 protein was previously known to be homologous to the 49 kDa component of the mitochondrial enzyme. We show that the 30.4 kDa corresponds to the N-terminal part while the 49 kDa subunit corresponds to the C-terminal portion of the bacterial protein. Thus, this bacterial protein represents a fusion of the two mitochondrial polypeptides suggesting that the two complex I genes arose from a single ancestor. Our results indicate that the 30.4 kDa and 49 kDa subunits are part of a structural and functional unit in complex I.

Complementary DNA sequences of the 24 kDa and 21 kDa subunits of complex I from Neurospora.

We have cloned and sequenced cDNAs coding for two subunits of the peripheral arm of Neurospora crassa complex I. The two polypeptides are synthesized as precursor proteins which are processed to mature forms with predicted molecular masses of 24331 and 20982 Da.

[Comparative study of transthoracic Doppler echocardiogram, computed tomography and aortography in the diagnosis of thoracic aortic dissection]. / Estudo comparativo do ecocardiograma Dopller transtorácico, tomografia computadorizada e aortografia no diagnóstico de dissecção da aorta torácica.

PURPOSE: Comparative and prospective evaluation of three methods (transthoracic echo-Doppler (TTE), computerized tomography (CT) and aortography (AORT) utilized for aortic dissection diagnosis. METHODS: The 39 patients with confirmed aortic dissection (surgery or autopsy) underwent, within a few hours of each other, all three methods scrutinized. There were 19 cases of type A and 20 of type B dissection. RESULTS: In type A dissection the methods were equivalent (TTE = 73.7%, CT = 84.2%, AORT = 73.7% p = NS) but for type B, TTE was significantly inferior to the other two methods (TTE = 60%, TC 90%, AORT = 80%, p < 0.05 for TTE, for TC and AORT p = NS). In three occasions, even though all three methods were performed, the diagnosis was not obtained. CONCLUSION: The methods which were evaluated make the diagnosis in the majority of cases. In type A all methods are similar, however, in type B, TC and AORT are superior to TTE. Even performing all three methods in each patient, in three instances the diagnosis was not made.

Characterization of a membrane fragment of respiratory chain complex I from Neurospora crassa. Insights on the topology of the ubiquinone-binding site.

1. A membrane fragment of complex I from the fungus Neurospora crassa was isolated by immunoprecipitation from alkaline-extracted mitochondrial membranes. 2. Analysis of the polypeptide composition of this hydrophobic domain of complex I has brought insights on the topology of two subunits of the enzyme, namely the 20.8 and 9.3 kDa components. 3. Our results indicate that the ubiquinone-binding site of complex I resides in the interface of the peripheral and membrane arms of the enzymes. The significance of these findings are discussed.

In organello assembly of respiratory-chain complex I: primary structure of the 14.8 kDa subunit of Neurospora crassa complex I.

A cDNA encoding the 14.8 kDa subunit of complex I from Neurospora crassa was cloned and sequenced. The deduced primary structure of this subunit reveals a predominantly hydrophilic protein containing no obvious membrane-spanning domain. In agreement with this characteristic, we have localized the 14.8 kDa subunit in the peripheral arm of the enzyme. The 14.8 kDa subunit was found to be conserved in mammalian complex I. The conservation of this subunit in such distantly related organisms suggests that the 14.8 kDa subunit is an important component of complex I. We have used an in organello system to study the biosynthetic pathway of this subunit. The 14.8 kDa polypeptide could be efficiently imported into isolated mitochondria. Furthermore, a fraction of the in-vitro-imported subunit was found to assemble in complex I. This is the first time that assembly in complex I of an in-vitro-synthesized subunit is demonstrated.

Cloning, in vitro mitochondrial import and membrane assembly of the 17.8 kDa subunit of complex I from Neurospora crassa.

We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of complex I from Neurospora crassa. The deduced primary structure of this subunit was partially confirmed by automated Edman degradation of the isolated polypeptide. The sequence data obtained indicate that the 17.8 kDa subunit is made as an extended precursor of 20.8 kDa. Resistance of the polypeptide to alkaline extraction from mitochondrial membranes and the existence of a putative membrane-spanning domain suggests that the 17.8 kDa subunit is an intrinsic (bitopic) membrane protein. The in vitro synthesized precursor of the 17.8 kDa subunit can be efficiently imported into isolated mitochondria, where it is cleaved to the mature species by the metal-dependent matrix-processing peptidase. The in vitro imported mature subunit is found mainly exposed to the mitochondrial intermembrane space. However, a significant fraction of the imported polypeptide acquires the same membrane topology as the endogenous subunit, indicating that correct assembly in the mitochondrial inner membrane did occur.

The 12.3 kDa subunit of complex I (respiratory-chain NADH dehydrogenase) from Neurospora crassa: cDNA cloning and chromosomal mapping of the gene.

The 12.3 kDa subunit of complex I (respiratory-chain NADH dehydrogenase) is a nuclear-coded protein of the hydrophobic fragment of the enzyme. We have isolated and sequenced a full-length cDNA clone coding for this polypeptide. The deduced protein is 104 amino acid residues long with a molecular mass of 12305 Da. This particular subunit of complex I lacks a cleavable mitochondrial targeting sequence. In agreement with its localization within complex I, we have found that this subunit behaves like an intrinsic membrane protein. Nevertheless, the deduced protein is rather hydrophilic, exhibiting no hydrophobic domain long enough to traverse a membrane in an alpha-helical conformation. The 12.3 kDa subunit shows a significant similarity to the hinge protein of complex III, suggesting that these two polypeptides may be involved in identical functions. This complex I subunit is coded for by a single gene. Applying restriction-fragment-length-polymorphism mapping, we located the gene on the right side of the centromere in linkage group I, linked to the lys-4 locus.

[Transesophageal echocardiography study with dynamic spontaneous contrast of the left atrial appendage, its relationship with the anatomy and thrombus formation]. / Estudo ecocardiográfico transesofágico do contraste espontâneo dinâmico no apêndice auricular esquerdo, sua relação com a anatomia e formação de trombos.

OBJECTIVE: Transesophageal two-dimensional echocardiographic study of anatomical characteristics of the left atrial appendage and its relation to spontaneous dynamic echocardiographic contrast. DESIGN: Outpatients undergoing a prospective two-dimensional transesophageal echocardiographic study. SETTING: Consecutive outpatients studied at the Echocardiographic Laboratory of Gregorio Marañon General Hospital, Madrid. MATERIAL AND METHODS: In each patient at the level of the left atrial appendage we calculated the following transesophageal echocardiographic parameters: end-systolic and end-diastolic maximal longitudinal and transversal diameters, total systolic and diastolic areas, percentage of systolic fractional shortening, presence of left atrial appendage thrombus and spontaneous dynamic echo-contrast. MAIN RESULTS: Left atrial appendage spontaneous dynamic contrast was observed in 48% of the total population. In the group of patients with left atrial spontaneous echo-contrast we observed larger longitudinal systolic (44 +/- 14 mm vs 28 +/- 13 mm, p = 0.01) and diastolic (52 +/- 16 mm vs 38 +/- 12 mm, p = 0.005) diameters, larger transversal systolic (25 +/- 10 mm vs 19 +/- 6 mm, p = 0.03) and diastolic (28 +/- 8 mm vs 25 +/- 9 mm, p = NS) diameters and also larger systolic (601 +/- 204 mm2 vs 337 +/- 110 mm2, p < 0.0001) and diastolic (715 +/- 230 mm2 vs 507 +/- 184 mm2, p = 0.001) areas, compared to the group without this dynamic echocardiographic phenomena. Left atrial appendage percentage of fractional shortening was considerably reduced in patients with spontaneous dynamic echo-contrast (15 +/- 14% vs 39 +/- 18%, p = 0.001) and related to local thrombus formation (13% vs 1%, p < 0.001). CONCLUSIONS: Left atrial spontaneous dynamic echo-contrast is more common in patients with enlarged left atrial appendage systo-diastolic diameters and areas. In this group of patients the presence of left atrial spontaneous echo-contrast is related to a significant reduction in left atrial appendage contractile function and thrombus formation. Parameter analysis of left atrial appendage anatomy by two-dimensional transesophageal echocardiography may have clinical relevance in the assessment of patients with high risk for left atria thromboembolic phenomena.

[Meaning of mosaic color area in mitral regurgitation. Echocardiographic transesophageal study]. / Significado da área de mosaico cor na regurgitação mitral. Estudo ecocardiográfico transesofágico.

OBJECTIVE: The purpose of our study was to analyse the meaning of total and mosaic color Doppler area of the mitral regurgitation jet, in terms of the degree of mitral regurgitation severity. PATIENTS: In and out patients referred to the Echocardiographic Laboratory of Gregorio Marañon General Hospital, Madrid. SETTING: Transesophageal echocardiographic prospective study MATERIAL AND METHODS: By pulsed and color Doppler transesophageal approach we studied 94 consecutive patients with mitral regurgitation diagnosis. We divided the entire population in three groups according to the degree of transthoracic mitral regurgitation severity and mitral regurgitation color area index (Groups I, II and III). In each patient we systematically measured the regurgitant maximal area (AT) and of the aliasing color area, as well as maximal peak velocity (VIS) and area (AIS) of the reversed pulmonary venous pulsed Doppler flow obtained at the level of the left upper pulmonary vein. RESULTS: For the group I, color Doppler AT was 411 +/- 315 mm2 and AN was 204 +/- 123 mm2 (R = 0.25), pulmonary venous pulsed Doppler VIS was 4 +/- 8 cm/sec (R = NS for AT and 0.79 for AN) and AIS was 9 +/- 6 mm2 (R = NS for AT and 0.82 for AN). In the group II, color Doppler AT was 802 +/- 447 mm2, AN was 671 +/- 307 mm2 (R = 0.42). the pulmonary venous pulsed Doppler VIS was 22 +/- 12 cm/sec (R = NS for AT and 0.66 for AN). In the group III we obtained an AT value of 1174 +/- 462 mm2 and an AN value of 1092 +/- 417 mm2 (R = 0.62). In this group the pulmonary venous pulsed Doppler VIS was 50 +/- 13 cm/sec (R = 0.57 for AT and 0.76 for AN) and the correspondent AIS was 671 +/- 570 mm2 (R = 0.38 for AT and 0.91 for AN). CONCLUSIONS: Mosaic transesophageal echocardiographic color Doppler area of mitral regurgitant jets has a direct relationship with the reversal criteria of pulsed Doppler pulmonary venous flow. This relationship is greater than the total color Doppler area of the same regurgitant jet. The mosaic color Doppler area of mitral regurgitant jets is a more correct estimation of the systolic variation of left atrial pressure, when compared with the total color area of mitral regurgitation.